Our studies have two basic studies. The first is to understand how the oligosaccharide units of glycoproteins are synthesized and how they function as recognition markers in biologic processes. The second is to understand at the molecular level how newly synthesized lysosomal enzymes are directed to lysosomes. For the first goal, we plan to use lectin- resistant cell lines with specific blocks in oligosaccharide biosynthesis to uncover new pathways for the synthesis of oligosaccharides. Several approaches will be made to understand lysosomal enzyme targeting. First, we plan to identify the common protein recognition marker for lysosomal enzymes required for the generation of phosphomannosyl residues which serve as the recognition signal for targeting proteins to lysosomes. To do this, we will construct chimeric molecules between the secretory protein pepsinogen and the lysosomal enzyme cathepsin D to define the regions on cathepsin D needed for recognition by phosphotransferase and subsequence phosphorylation. We plan to clone phosphotransferase and to use this clone to study the defects in patients with mucolipidosis II and III, two autosomal recessive genetic disorders of phosphotransferase. We also plan to clone alpha N-acetylglucosamine-1-phosphodiester N- acetylglucosaminidase, the second enzyme required for the generation of phosphomannosyl residues. In addition, we plan to mutagenize the cDNA's for the cation-independent and the cation-dependent mannose 6-phosphate receptors to generate receptor molecules with mutations and deletions in their cytoplasmic domains. These cDNA's will be transfected into receptor negative mouse L cells to determine the effects on receptor function in lysosomal enzyme sorting at the Golgi and endocytosis at the cell surface.